Biocatalytic Cascades toward Iminosugar Scaffolds Reveal Promiscuous Activity of Shikimate Dehydrogenases

Iminosugar scaffolds are highly sought-after pharmaceutical targets, but their chemical synthesis is lengthy and can suffer from poor scalability and purification. Here we report protecting-group-free chemoenzymatic and biocatalytic cascades to synthesize iminosugars from sugar-derived aminopolyols in two steps. Using galactose oxidase variant F2 followed by a chemical or enzymatic reduction provided an efficient one-pot route to these targets, with product formation >70%. Key to success of this strategy was the application of genome mining, which identified bacterial shikimate dehydrogenases as promiscuous iminosugar reductases. The cell-free protocols allowed for isolation of highly polar iminosugar products from biotransformations in a single step through development of a gradient-elution cation exchange purification. The two-step pathway provides a short synthetic route that can be used as a cell-free platform for broader iminosugar synthesis.


Materials and methods
All commercial reagents and solvents used in this work were purchased from Sigma-Aldrich (Poole, Dorset,UK), Alfa-Aesar (Heysham, Lancashire, UK), Acros Organics (Loughborough, UK), Santa Cruz Biotechnology (Dallas, Texas, U.S.A.) or Fluorochem (Hadfield, Derbyshire, UK) and used without further purification. Isopropyl β-D1-thiogalactopyranoside (IPTG), kanamycin, Terrific broth (TB) and LB (Luria-Bertani) Agar Miller was purchased from Formedium (Hunstanton, England). Co-factors were obtained from Prozomix Ltd (Haltwhistle, Northumberland, UK). Unless stated otherwise, a Bruker Avance 400 MHz spectrometer was used to record NMR spectra with chemical shifts reported in ppm relative to tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Reverse phase UPLC-QDa analysis was carried out on a Waters Acquity H-class system with sample manager (model J15SD1368G), column heater (model H15CHA), FLR detector (Model G15UPF045G), TUV detector (model E16TUV487A), QDa detector (model KAB1525). A HSS C18 1.8 μm, 2.1 x 100 mm column was used as stationary phase. Normal phase HILIC UPLC-QDa was carried out on the same instrument using a Glycan BEH amide 130 Å, 1.7 μm, 2.1 x 150 mm column as stationary phase. Thompson UPLC grade nano filter vials with PVDF membrane, 0.45 μm pore size were used for all samples and standards. Reverse phase UPLC-QDa was applied for all biotransformations of 1-amino-2-deoxy-D-ribitol 1 and related standards. Normal phase HILIC UPLC-QDa was used for standards and biotransformations of all other substrates.

AQC derivatisation
Stocks of the AQC-tag reagent (6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate) were made up at 10 mM in dry acetonitrile and stored at -80 o C as single use aliquots.
For analysis by normal phase HILIC UPLC, the above procedure was followed except borate buffer was replaced by an equal volume of 1:1 acetonitrile/borate buffer (100 mM, pH 8.8).
Enzyme immobilisation Immobilised GOase F 2 was prepared as described in the literature. 1 Purolite butylmethacrylate ECR8285 resin (10-20 mg) was washed with NaPi/NaCl buffer (25 mM NaPi, 100 mM NaCl, 3 x 1 mL). GOase F 2 (10 % w/w, 1-2 mg) was added and the solution made up to 1 mL with NaPi/NaCl buffer. The suspension was gently agitated at ambient temperature for 16 h then allowed to stand for a further 24 h. The beads were washed with reaction buffer (typically 100 mM NaPi pH 7.4) and stored at 4 o C until use. Immobilisation yields were monitored by Nanodrop absorbance or BSA assay, and were comparable to previous reports.

Biocatalyst Production
Chemically competent E. coli cells were transformed with DNA plasmid vectors that contain genes for the desired protein (biocatalyst) and grown on LB agar plates containing 30 µg mL -1 antibiotic (kanamycin) as shown in Table S1. Galactose oxidase (GOase) production A single colony harbouring a GOase gene was selected and used to inoculate 5 mL of lysogeny broth (LB) medium containing kanamycin (final concentration 30-50 μg mL -1 ) in a 15 mL falcon tube and incubated overnight at 37 o C shaking at 250 rpm. 500 μL of the preculture was used to inoculate a 250 mL of auto induction medium (8ZY-4LAC) supplemented with 250 μL of kanamycin (30 mg mL -1 ) in a 2 L baffled flask and further incubated at 250 rpm at 26 o C for 60 h. The cell cultures were harvested by centrifugation at 4000 rpm for 30 min at 4 o C and the supernatant was discarded. The cell pellets were washed with NaPi buffer (100 mM, pH 7.4) and centrifuged again using the same conditions and the supernatant discarded. The wet cell pellets were stored at -20 o C. To purify the protein the thawed cell pellets of one 400 mL culture were first resuspended in NP buffer (50 mM Na 2 PO 4 , 100mM NaCl, pH 8.0, 25 mL) with Triton X 100 lysis buffer (250 uL) and lysozyme from egg white (1 mg mL -1 ) and incubated at 4 o C for 20 min with gradual shaking. The suspension was lysed by ultra-sonification (30 sec ON, 90 sec OFF, 5x cycles) while submerged in an ice bath. The lysate was clarified by centrifugation at 20,000 rpm for 40 min at 4 o C. The supernatant was then loaded onto a Strep-TagII® column which had been equilibrated with 50 mL NP Buffer and the column was subsequently washed with 30 mL NP buffer to remove any non-tagged proteins. The GOase was eluted with 40 mL NPD buffer (50 mM Na2PO4, 300 mM NaCl, 5 mM desthiobiotin, pH 8) and concentrated by centrifugation in 30,000 MWCO PES vivaspin columns. The protein was dialysed overnight at 4 o C against 50 mM NaPi (pH 7.4) supplemented with copper sulphate. After, the protein was dialysed again at 4 o C against NaPi buffer to remove excess Cu. The protein concentration was then determined using a NanoDrop TM spectrometer (typically 3-5 mg ml -1 ). The samples were divided into 1.5 mL Eppendorf tubes, flash-frozen and then stored at -80 o C until they were used in the biotransformation reactions. Typical expression yields for GOase variants were 200 -250 mg/L culture broth, in line with previous reports. 2 Reductase production S5 A single colony harbouring a reductase gene was added to 20 mL LB medium supplemented with 20 μL kanamycin (30 mg mL -1 ) and incubated overnight at 250 rpm and 30 o C. The full volume of this preculture was used to inoculate a 400 mL of TB media, supplemented with 400 μL of kanamycin, in a 2 L baffled flask and further incubated at 37 o C, 250 rpm until optical cell density (OD600) of 0.6 was reached. At this point, protein expression was induced by inoculating the flask with 400 μL of isopropyl-beta-D-1-thiogalactopyranoside (IPTG, 0.1 M). The flask was then incubated at 22 o C at 200 rpm for 20 h. The cells were harvested by centrifugation at 4000 rpm for 30 min at 4 o C. The cell pellets were washed with NaPi buffer (100 mM, pH 7.4) and centrifuged again using the same conditions and the supernatant discarded. The cell pellets were stored at -20 o C in 50 mL falcon tubes until further use. For preparation of cell free extracts, the thawed cell pellets were resuspended in 4-5 mL (per g of cell pellet) in NaPi buffer (100 mM, pH 7.4) and lysed by ultra-sonication (60 sec ON, 120 sec OFF, 40 AMP, 4 cycles) while samples were submerged in an ice bath. The lysed cells were clarified by ultra-centrifugation at 18,000 rpm at 4 o C for 60 min. The clarified supernatant was filtered through a cellulose membrane (0.45 μm), frozen at -80 o C then lyophilised on a Buchi Lyovapor-200. Lyophilised cell free extract was stored at -20 o C until further use.

Reductase purification
Thawed cell pellets were resuspended in 4-5 mL (per g of cell pellet) in 10 % Buffer B (100 mM NaPi buffer, 300 mM NaCl, 30 mM imidazole, pH 7.5) and lysed by ultra-sonication (60 sec ON, 120 sec OFF, 40 AMP, 4 cycles) while samples were submerged in an ice bath. The lysed cells were clarified by ultra-centrifugation at 18,000 rpm at 4 o C for 60 min. The clarified supernatant was filtered through a cellulose membrane (0.45 μm) and loaded onto a His-Trap Crude FF column (GE Healthcare) charged with 0.1 M nickel sulphate equilibrated with 10% buffer B. The column was washed with 15 mL 10% buffer B and then 15 mL 20% buffer B. His-tagged protein was then eluted with 100% buffer B and 1-2 mL fractions were collected. The protein concentration of each fraction was monitored by a thermofisher NanoDrop TM microvolume spectrophotometer. Fractions containing pure protein were combined and then concentrated using a membrane 30,000 MWCO PES vivaspin columns to remove excess imidazole and exchange into NaPi buffer until the desired concentration was achieved (typically 10 mg mL -1 ). The pure protein was divided into 1 mL aliquots, flash frozen and stored at -80 o C until further use. Expression yields of reductase proteins were between 100-150 mg/L of culture broth.

SDS-PAGE analysis
SDS PAGE was carried out on BioRad premade gel in 1X TGS running buffer. Samples were loaded with 50% Lameli buffer purchased from Sigma Aldrich, Molecular weight marker was purchased from New England Biolabs.

Identification of new putative reductases
Multigeneblast searches were performed on two gene clusters that have been identified to produce iminosugars. [3][4][5] Four searches were carried out, two using the reported biosynthetic gene cluster sequences and two using those sequences followed by the sequence of a putative IRED from B. Amyloli and C. pinensis. In total, 45 potential reductases were identified, which were trimmed down to a final 15. A multiple sequence alignment was carried out and a phylogenetic tree was generated, demonstrating the broad scope of the enzymes mined. It was our intention to generate a very diverse panel of possible reductases, as many enzymes discovered that carry out imine reductions or reductive animations are often mislabelled or part of other superfamilies. 6 One of these 15 enzymes had previously been reported to catalyse the endocyclic imine reduction of a 2-methylpiperideine. 7 The sequence of 7 homologs were also disclosed as putative IREDs and so were also investigated for our cascade.
Once pRed-14 shikimate dehydrogenase had been identified as a positive hit in our cascade, a further 7 homologs of this protein were also identified and ordered for further testing. Colourimetric reductase screening A colourimetric IRED screen was carried out in the oxidative direction as described in the literature using cis-3,4-dihydroxypiperidine as the substrate. 6 Assay plates were prepared using cell free extracts of the putative reductases identified in this work. Each well contained 0.25 mg mL -1 INT, 0.5 mM NAD(P) + , 0.75 % v/v diaphorase, 4 mg mL -1 reductase lysate and 10 mM substrate, made up to a total volume of 200 μL with 100 mM Tris.HCl pH 9.0. Absorbance at 490 nm was measured before and after incubation at ambient temperature for 24h. After incubation, a deep red colour was observed in almost all wells including blanks and no meaningful conclusions could be drawn. Due to this and the lack of availability of the cascade intermediates, the reductases were screened in the context of the full cascade. When later validated in forward direction biotransformations, almost all of these results were shown to be false positives, i.e. not indicative of activity towards the desired imine reduction. Similar results have been observed with other iminosugar scaffolds and this colourimetric assay (data not shown here), possibly due to alcohol dehydrogenase activity present in endogenous enzymes within the lysate, or of the putative reductases.  GOase activity was initially determined by the well-known HRP-ABTS assay, where ABTS is reduced by HRP and H 2 O 2 generated during oxidation of the substrate. 1,8 In triplicate, purified GOase enzyme (10 μL, 0.4-0.5 mg mL -1 stock) was supplemented with 90 μL of a reaction mix containing HRP (0.23 mg mL-1) and ABTS (0.4 mg mL -1 ) in NaPi buffer (100 mM) in a 96 well plate. The assay reaction was initiated by addition of a substrate solution (100 μL, 50 mM in NaPi buffer) and absorbance was measured at 420 nm for 10 minutes and specific activities were calculated in μmol min -1 mg -1 using the following formula.
, and is the initial rate of change in = final enzyme concentration ∆ absorbance at 420 nm. The oxidation of 1 mol alcohol produces 1 mol of H 2 O 2 but releases 2 mol ewhich reduce 2 mol ABTS, therefore . = 2 Table S4: Specific activity of GOase variants against the aminopolyol substrate panel, measured using the above procedure. n.t. = not tested, blank entries where no activity was observed.
GOase variant specific activity / μmol Assay to determine inhibition of GOase by iminosugar products The HRP-ABTS assay was also used to determine if the iminosugar products formed in our cascade would inhibit GOase F 2 . The assay was carried out as above, using 1,2,4-butanetriol and glycerol as model substrates and adding increasing concentrations of cis-3,4-dihydroxy piperidine to the reaction mix. Calculation of specific activities showed no significant reduction in activity in the presence of cis-3,4-dihydroxy piperidine. Using the same assay, it was determined that GOase F 2 exhibits no activity for cis-3,4-dihydroxy piperidine. Concentration of 3,4-dihydroxypiperidine / mM Specific activity / umol/min.mg Figure S4: Specific activities of GOase F 2 against 1,2,4-butane triol and glycerol with increasing concentration of cis-3,4-dihydroxy piperidine.

Biotransformation Methods Chemoenzymatic GOase-NaCNBH 3 cascade biotransformations
Reactions were carried out in 2 mL Eppendorf tubes with a total reaction volume of 250 μL. Each reaction contained components diluted from stock solutions, with final concentrations of 5 mM amino alcohol substrate, 1 mg ml -1 purified GOase, 0.1 mg ml -1 catalase, 0.1 mg ml -1 HRP, in 100 mM NaPi pH 7.4. Reactions were incubated at 30 o C, 200 rpm for 6 h followed by addition of NaCNBH 3 (25 mM, 5 equiv.) and incubation at 30 o C, 200 rpm for 16 h. After incubation, the biotransformations were quenched with an equal volume of MeOH and centrifuged (13k rpm, 10 minutes). The supernatant was decanted and derivatised using the AQC-tag procedure outlined above for analysis by reverse phase UPLC-QDA.
Screening of proposed GOase-Reductase cascade 1-amino-2-deoxy-D-ribose was used as a model substrate for investigations into the proposed GOase-reductase cascade.
UPLC-QDA analysis of the tagged reaction mixtures revealed low levels of product formation (< 5%) with two of the enzymes tested. Several side product peaks were also observed and could be due to GOase catalysed oxidation of the glucose present in the reaction mix or side reactions of endogenous proteins remaining in the cell free extract. As such, further screening reactions were carried out using purified protein and stoichiometric NAD(P)H instead of NAD(P) + recycling. One-pot biotransformations were carried out with either simultaneous or sequential addition of GOase and Reductase components. Reaction mixes were prepared in NaPi buffer (100 mM, pH 7.4) at a total volume of 250 μL and typically contained; purified GOase F 2 (1 mg mL -1 ), HRP (0.1 mg mL -1 ), catalase (0.1 mg mL -1 ), and 1-amino-2-deoxy-D-ribose (5 mM). Simultaneous addition biotransformations were supplemented with purified pRed enzyme (1 mg mL -1 final) and NAD(P)H (1 equiv., 5 mM) before incubation at 25-30 o C for 16 hours. In sequential addition biotransformations, the GOase reaction was incubated for 3-6 hours before addition of purified pRed enzyme (1 mg mL -1 final) and NADH (1 equiv., 5 mM) and incubation at 25-30 o C for 16 hours. After incubation, biotransformations were quenched with methanol (250 μL) and centrifuged (13k rpm, 10 min) before derivatisation with the AQC-tag and UPLC-QDA analysis. Figure S5: Reverse phase UPLC-QDA traces of biotransformations with purified pRed-14 and 1amino-2-deoxy-ribitol. Left: simultaneous addition, 7% product formation. Right: sequential addition of pRed/co-factor after 3 h,19% product formation.

Investigations into reaction pH
Reactions were performed as above in various pH of NaPi buffer either from start to finish or by beginning the reaction at one pH and adjusting upon addition of the reductase components. Reactions were incubated at 30 o C. The following data shows that the one-pot cascade was most efficient when pH was maintained at 8.0 throughout.

Investigations into reaction temperature
Reactions were performed as above (in NaPi buffer pH 7.4) with either 5 or 10mM substrate and incubated at 25 or 30 o C. It was observed that the lower reaction temperature improved overall product formation, likely due to an improvement in enzyme stability. A small drop in formation of product was observed from 5 to 10 mM substrate at both temperatures.

Investigations into substrate loading
Reactions were performed as above using a range of substrate concentrations with both free and immobilised GOase F 2 in NaPi (100 mM, pH 8.0), incubated at 25 o C. Generally, as substrate concentrations increased the formation of product decreased, although for immobilised GOase a higher product formation was seen for 25 mM substrate than with 10 mM. A large decrease in product formation was observed upon increasing substrate concentration to 50 mM with both free and immobilised GOase ( Figure S7 & S8, respectively). 5 10
Pleasingly, implementation of either GDH or PtDH recycling systems improved product formation when compared to stoichiometric NADH. The slightly better performance of PtDH recycling system may relate to elimination of glucose in the reaction mixture acting as a competing substrate for GOase F 2 , thus reducing side reactions and GOase deactivation. Negative control reactions containing no pRed-14 were also performed and showed that the GDH/ PtDH recycling enzymes did not provide any product formation. Optimisation of NAD + co-factor recycling Reactions were performed as above using free GOase F 2 and 10 mM substrate in NaPi buffer pH 8.0, incubated at 25 o C. Biotransformations were performed in one-pot sequential addition mode and reaction parameters were varied as follows. Reaction mixes were prepared in NaPi buffer (10-100 mM, pH 8) at a total volume of 250 μL and typically contained; purified GOase F 2 (1 mg mL -1 ), HRP (0.1 mg mL -1 ), catalase (0.1 mg mL -1 ), and 1-amino-2-deoxy-D-ribose (10 mM). The reaction was incubated for 4 hours before addition of purified pRed enzyme (0.25-1 mg mL -1 final), NAD (0.5 mM), PtDH lysate (0.05-0.3 mg mL -1 ) and NaPt (10-75 mM final) and incubation at 25 o C for 16 hours. After incubation, biotransformations were quenched with methanol (250 μL) and centrifuged (13k rpm, 10 min) before derivatisation with the AQC-tag and UPLC-QDA analysis. Biotransformations were performed in one-pot sequential addition mode with the optimised conditions. Reaction mixes were prepared in NaPi buffer (10 mM, pH 8) at a total volume of 250 μL and contained; purified GOase F 2 (1 mg mL -1 ), HRP (0.1 mg mL -1 ), catalase (0.1 mg mL -1 ), and substrate 1-7 (10 mM). The reaction was incubated for 6 hours before addition of purified reductase enzyme (0.5 mg mL -1 final), NAD + (0.5 mM), PtDH lysate (0.2 mg mL -1 ) and NaPt (50 mM final from a 1M stock at pH 8.0) and incubation at 25 o C for 16 hours. After incubation, biotransformations were quenched with methanol (250 μL) and centrifuged (13k rpm, 10 min) before derivatisation with the AQC-tag and UPLC-QDa analysis. These reactions were performed with MGB14 and two homologs, YdiB and AroE shikimate dehydrogenases from E. coli.
Select reactions were scaled up to 10 mL in order to characterise products by NMR.

Purification procedure:
Crude reaction mixtures with phosphate salts and protein removed were concentrated and resuspended in minimal H 2 O then loaded onto a pre-washed column of Dowex 50WX8 H + or NH 4 + form. The loaded column was washed once more with H 2 O (3-5 cv.) and then fractions were collected with an increasing concentration of aqueous NH 3 (0-1 M, 50 mM increments, 3 cv. per solution). The fractions were analysed by TLC or UPLC-QDa, concentrated in vacuo, resuspended in 1M HCl and concentrated in vacuo once more. Using this approach it was possible to separate the linear amino alcohol substrate from the cyclic iminosugar product and obtain pure compound for characterisation. In select cases where fractions were mixed or not completely pure, the above procedure was applied again using a column of Dowex 50WX8 in NH 4 + form.

Deuteration experiments used to investigate reduction mechanism
Biotransformations were performed in one-pot sequential addition mode with a deuterium based co-factor recycling system. Reaction mixes were prepared in H 2 O or D 2 O at a total volume of 1 mL and contained; GOase F 2 (20 mg, 10% w/w, immobilised on Purolite ECR8285 epoxy), HRP (0.1 mg mL -1 ), catalase (0.1 mg mL -1 ), and substrate 1-7 (10 mM, from a stock in H 2 O or D 2 O). The reaction was incubated for 6 hours before addition of purified reductase enzyme (0.5 mg mL -1 final), NAD + (0.5 mM), CDX-901 GDH lysate (0.2 mg mL -1 ) and D-glucose-d1 (50 mM final) and incubation at 25 o C for 16 hours. After incubation, biotransformations were centrifuged (13k rpm, 10 min) before derivatisation with the AQC-tag and UPLC-QDa analysis.        Figure S27: Example reverse phase (C18) UPLC-QDa trace of an AQC-tagged biotransformation with 1-amino-2-deoxy-ribitol 1, GOase F 2 and NaCNBH 3 . The by-product peak at 1.718 is assumed to be the 3,4-trans diol diastereomer of the piperidine product, which could be generated by non-selective reduction of a racemised intermediated generated through imine-enamine tautomerisation. Importantly, no such by-product peak is observed in any of the biocatalytic reductase reactions.

N-methyl-3,4,5,6-tetrahydroxyazepane 7a
A 10 mL biocatalytic cascade reaction with substrate 7 (10 mM), immobilised GOase F 2 (100 mg, 10 w% enzyme loading), HRP (0.1 mg mL -1 ) and catalase (0.1 mg mL -1 ) in NaPi buffer (100 mM, pH 8.0) was incubated at 25 o C for 6 hours. The reaction was then supplemented with NaCNBH 3 (50 mM) and incubated at 25 o C for a further 16 hours. CaCl 2 was added to precipitate phosphate salts and the suspension was filtered (30K MWCO) and lyophilised. The residue was resuspended in minimal water and loaded on to a column of Dowex 50WX8 H + form resin (prewashed with water). The loaded column was washed once more with H 2 O (3-5 cv.) and then fractions were collected with an increasing concentration of aqueous NH 3 (0-1 M, 50 mM increments, 3 cv. per solution). Fractions containing product (as determined by UPLC-QDa) were pooled and concentrated in vacuo to afford the title compound 7a as a yellow oil (13 mg, 73%    Figure S41: 13 C-NMR spectra of N-methyl-3,4,5,6-tetrahydroxyazepane 7a synthesised through the GOase-NaCNBH 3 cascade. To a solution of 2-deoxyribose (536 mg, 4 mmol) in a saturated solution of NH 4 OAc in EtOH (80 mL) were added NaCNBH 3 (744 mg, 12 mmol) and 30% aq. NH 3 (32 mL). The mixture was stirred at reflux for 18 h, cooled to room temperature and concentrated under reduced pressure. The residue was redissolved in water (10 mL, loaded on to a column of Dowex ion-exchange resin (prewashed with water), and washed with water (100 mL) to remove excess salt. The amine product was then eluted with 30% aq NH 3 (30 mL). The eluent was concentrated under reduced pressure, resuspended in HCl (1 mL, 1M) and concentrated again to afford the title compound as an off-white oil (399 mg, 73% To a solution of D-xylose (150 mg, 1 mmol) in a saturated solution of NH 4 OAc in EtOH (20 mL) were added NaCNBH 3 (188 mg, 3 mmol) and 30% aq. NH 3 (8 mL). The mixture was stirred at reflux for 18 h, cooled to room temperature and concentrated under reduced pressure. The residue was redissolved in water (5 mL, loaded on to Dowex ion-exchange resin (pre-washed with water), and washed with water (100 mL) to remove excess salt. The amine product was then eluted with 30% aq. NH 3 (30 mL). The eluent was concentrated under reduced pressure, resuspended in HCl (1 mL, 1M) and concentrated again to afford the title compound as an offwhite solid (78 mg, 51% To a solution of D-arabinose (150 mg, 1 mmol) in a saturated solution of NH 4 OAc in EtOH (20 mL) were added NaCNBH 3 (188 mg, 3 mmol) and 30% aq. NH 3 (8 mL). The mixture was stirred at reflux for 18 h, cooled to room temperature and concentrated under reduced pressure. The residue was redissolved in water (5 mL, loaded on to Dowex ion-exchange resin (pre-washed with water), and washed with water (100 mL) to remove excess salt. The amine product was then eluted with 30% aq. NH 3 (30 mL). The eluent was concentrated under reduced pressure, resuspended in HCl (1 mL, 1M) and concentrated again to afford the title compound as an offwhite solid (45 mg, 29%). δH To a stirred solution of glucosamine.HCl (1.0 g, 4.64 mmol) in water (20 mL) was added NaBH 4 (263 mg, 6.96 mmol, 1.5 eq.) in water (10 mL). The solution was stirred at room temperature for 18 h, quenched with conc. HCl and concentrated under reduced pressure. The resulting yellow oil was redissolved in water (10 mL), loaded onto a column of Dowex ion-exchange resin (50WX8, prewashed with water) and washed with water (100 mL). The amine product was then eluted with 30% aq. NH 3 (50 mL) and concentrated under reduced pressure to afford the title compound as a yellow solid (900 mg, 89% To a stirred solution of galactosamine.HCl (1.0 g, 4.64 mmol) in water (20 mL) was added NaBH 4 (263 mg, 6.96 mmol, 1.5 eq.) in water (10 mL). The solution was stirred at room temperature for 18 h, quenched with conc. HCl and concentrated under reduced pressure. The resulting yellow oil was redissolved in water (10 mL), loaded onto a column of Dowex ion-exchange resin (prewashed with water) and washed with water (100 mL). The amine product was then eluted with 30% aq. NH 3 (50 mL), concentrated under reduced pressure, resuspended in HCl (1 mL, 1M) and concentrated again to afford the title compound as a yellow syrup (590 mg, 58%). δH